Site-specific proteolytic processing plays a significant role in the regulation of cellular processes as diverse as signal transduction, RNA transcription , apoptosis, and development. In addition, specific processing of viral polypeptides is a critical stage in the replication and maturation of infectious particles. Although many general proteases have been identified, few of the enzymes responsible for site-specific processing events have been isolated. Therefore, a genetic screen has been developed that provides a rapid methodology for the isolation and characterization of these enzymes. The system is based on the highly characterized bacteriophage lambda lytic-lysogenic cycle. Expression of the phage-encoded repressor results in repression of the bacteriophage's lytic functions. Induction from the lysogenic state is initiated by specific cleavage of the repressor, resulting in the expression of the phage's replicative functions and the lysis of the host cell. Using the HIV protease as a model enzyme, the developed system was shown to be capable of isolating a rare HIV protease-encoding phage from a complex phage pool. For the human immunodeficiency virus (HIV) the encoded protease is required for the replication of the virus and has been the target of novel anti-viral therapeutics. However, arising inhibitor resistant viral strains have become an increasingly significant clinical problem. Using the developed genetic selection, a bank of inhibitor-resistant mutants in this protease have been isolated including mutations which correlate with resistant clinical isolates. The rapid selection of such mutations has implications for the prediction of relevant mutations and may be applicable to other viral systems.